Pharma[e]cology: How the Gut Microbiome Contributes to Variations in Drug Response.

Drugs represent our first, and sometimes last, line of defense for many diseases, yet despite decades of research we still do not fully understand why a given drug works in one patient and fails in the next. The human gut microbiome is one of the missing puzzle pieces, due to its ability to parallel and extend host pathways for drug metabolism, along with more complex host-microbiome interactions. Herein, we focus on the well-established links between the gut microbiome and drugs for heart disease and cancer, plus emerging data on neurological disease. We highlight the interdisciplinary methods that are available and how they can be used to address major remaining knowledge gaps, including the consequences of microbial drug metabolism for treatment outcomes. Continued progress in this area promises fundamental biological insights into humans and their associated microbial communities and strategies for leveraging the microbiome to improve the practice of medicine.

Isothermal titration calorimetry and surface plasmon resonance methods to probe protein-protein interactions.

Isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) are two commonly used methods to probe biomolecular interactions. ITC can provide information about the binding affinity, stoichiometry, changes in Gibbs free energy, enthalpy, entropy, and heat capacity upon binding. SPR can provide information about the association and dissociation kinetics, binding affinity, and stoichiometry. Both methods can determine the nature of protein-protein interactions and help understand the physicochemical principles underlying complex biochemical pathways and communication networks. This methods article discusses the practical knowledge of how to set up and troubleshoot these two experiments with some examples.

Cell-Type Specificity of Mosaic Chromosome 1q Gain Resolved by snRNA-seq in a Case of Epilepsy With Hyaline Protoplasmic Astrocytopathy.

Objectives: Mosaic gain of chromosome 1q (chr1q) has been associated with malformation of cortical development (MCD) and epilepsy. Hyaline protoplasmic astrocytopathy (HPA) is a rare neuropathologic finding seen in cases of epilepsy with MCD. The cell-type specificity of mosaic chr1q gain in the brain and the molecular signatures of HPA are unknown. Methods: We present the case of a child with pharmacoresistant epilepsy who underwent epileptic focus resections at age 3 and 5 years and was found to have mosaic chr1q gain and HPA. We performed single-nuclei RNA sequencing (snRNA-seq) of brain tissue from the second resection. Results: snRNA-seq showed increased expression of chr1q genes specifically in subsets of neurons and astrocytes. Differentially expressed genes associated with inferred chr1q gain included AKT3 and genes associated with cell adhesion or migration. A subpopulation of astrocytes demonstrated marked enrichment for synapse-associated transcripts, possibly linked to the astrocytic inclusions observed in HPA. Discussion: snRNA-seq may be used to infer the cell-type specificity of mosaic chromosomal copy number changes and identify associated gene expression alterations, which in the case of chr1q gain may involve aberrations in cell migration. Future studies using spatial profiling could yield further insights on the molecular signatures of HPA.

Cell type specificity of mosaic chromosome 1q gain resolved by snRNA-seq in a case of epilepsy with hyaline protoplasmic astrocytopathy.

Introduction: Mosaic gain of chromosome 1q (chr1q) has been associated with malformation of cortical development (MCD) and epilepsy. Hyaline protoplasmic astrocytopathy (HPA) is a rare neuropathological finding seen in cases of epilepsy with MCD. The cell-type specificity of mosaic chr1q gain in the brain and the molecular signatures of HPA are unknown. Methods: We present a child with pharmacoresistant epilepsy who underwent epileptic focus resections at age 3 and 5 years and was found to have mosaic chr1q gain and HPA. We performed single-nuclei RNA-sequencing (snRNA-seq) of brain tissue from the second resection. Results: snRNA-seq showed increased expression of chr1q genes specifically in subsets of neurons and astrocytes. Differentially expressed genes associated with inferred chr1q gain included AKT3 and genes associated with cell adhesion or migration. A subpopulation of astrocytes demonstrated marked enrichment for synapse-associated transcripts, possibly linked to the astrocytic inclusions observed in HPA. Discussion: snRNA-seq may be used to infer the cell type-specificity of mosaic chromosomal copy number changes and identify associated gene expression alterations, which in the case of chr1q gain may involve aberrations in cell migration. Future studies using spatial profiling could yield further insights on the molecular signatures of HPA.

Expansion of a bacterial operon during cancer treatment ameliorates drug toxicity.

Dose-limiting toxicities remain a major barrier to drug development and therapy, revealing the limited predictive power of human genetics. Herein, we demonstrate the utility of a more comprehensive approach to studying drug toxicity through longitudinal study of the human gut microbiome during colorectal cancer (CRC) treatment (NCT04054908) coupled to cell culture and mouse experiments. 16S rRNA gene and metagenomic sequencing revealed significant shifts in gut microbial community structure during treatment with oral fluoropyrimidines, which was validated in an independent cohort. Gene abundance was also markedly changed by oral fluoropyrimidines, including an enrichment for the preTA operon, which is sufficient for the inactivation of active metabolite 5-fluorouracil (5-FU). Higher levels of preTA led to increased 5-FU depletion by the gut microbiota grown ex vivo. Germ-free and antibiotic-treated mice had increased fluoropyrimidine toxicity, which was rescued by colonization with the mouse gut microbiota, preTA+ E. coli, or CRC patient stool with high preTA levels. preTA abundance was negatively associated with patient toxicities. Together, these data support a causal, clinically relevant interaction between a human gut bacterial operon and the dose-limiting side effects of cancer treatment. Our approach is generalizable to other drugs, including cancer immunotherapies, and provides valuable insights into host-microbiome interactions in the context of disease.

Phage Immunoprecipitation-Sequencing Reveals CDHR5 Autoantibodies in Select Patients With Interstitial Lung Disease.

OBJECTIVE: Interstitial lung diseases (ILDs) are a heterogeneous group of disorders that can develop in patients with connective tissue diseases. Establishing autoimmunity in ILD impacts prognosis and treatment. Patients with ILD are screened for autoimmunity by measuring antinuclear autoantibodies, rheumatoid factors, and other nonspecific tests. However, this approach may miss autoimmunity that manifests as autoantibodies to tissue antigens not previously defined in ILD. METHODS: We use Phage Immunoprecipitation-Sequencing (PhIP-Seq) to conduct an autoantibody discovery screen of patients with ILD and controls. We screened for novel autoantigen candidates using PhIP-Seq. We next developed a radio-labeled binding assay and validated the leading candidate in 398 patients with ILD recruited from two academic medical centers and 138 blood bank individuals that formed our reference cohort. RESULTS: PhIP-Seq identified 17 novel autoreactive targets, and machine learning classifiers derived from these targets discriminated ILD serum from controls. Among the 17 candidates, we validated CDHR5 and found CDHR5 autoantibodies in patients with rheumatologic disorders and importantly, patients not previously diagnosed with autoimmunity. Using survival and transplant free-survival data available from one of the two centers, patients with CDHR5 autoantibodies showed worse survival compared with other patients with connective tissue disease ILD. CONCLUSION: We used PhIP-Seq to define a novel CDHR5 autoantibody in a subset of select patients with ILD. Our data complement a recent study showing polymorphisms in the CDHR5-IRF7 gene locus strongly associated with titer of anticentromere antibodies in systemic sclerosis, creating a growing body of evidence suggesting a link between CDHR5 and autoimmunity.